T helper (Th) cells in lymphoid tissues, depending on their location, develop into various Th subsets to regulate inflammation and defense. In the intestinal mucosa, fheir dominant phenotype is that of regulatory T (Treg) cells. Mucosal adjuvants, by overriding the tolerizing impact of mucosal Treg cells, are unique tools for dissecting immune regulation and inflammafion. Our preliminary data indicate that cholera toxin (CT) and related compounds induce Thi7 cell differentiation via a cAMP-dependent and IL-6/TGF(3-independent pathway. To elucidate the mechanism by which adenylyl cyclase toxins and other luminal microbial products (TLR ligands) stimulate and modulate the mucosal microenvironment to promote effector Thi7 responses, we propose in Aim 1 to define how oral CT administrafion enhances Th17-promoting dendritic cells (DC) and suppresses Treg-promoting DC. We will explore the role of cAMP and retinoic acid in these processes, and characterize the subsequent Th cell response provoked by different MLN DC subsets. In Aim 2, we will characterize the phenotype, stability and lineage differentiation markers of CD4 T cells from in vitro- and in vivo-differentiated CT-induced Thi7 subsets, and evaluate the infiammatory/regulatory impact of orally delivered CT on the outcome of Thi7 cell-mediated colitis. Aim 3 will explore the role of CGRPP produced by CT-activated DC in a mucosal Thi7 response. Aim 4 will investigate the potential contributing role of TLR stimulation to CT-induced Thi7 differentiation. Dissecting the impact of CT and other cAMP-inducing compounds on mucosal DC maturation will significantiy broaden our knowledge on the generation of mucosal Th responses. By dissecting the lineage stability of CT-induced mucosal Thi7 cells, their effector function, lineage commitment, trafficking and their interactions with other Th cells, we will increase our understanding of the regulation of inflammation and immunity at mucosal sites. The data generated in these studies may help to design new therapeutic approaches for mucosal inflammatory diseases and novel mucosal adjuvants. RELEVANCE (See instmctions): Mucosal adjuvants are critical for success of oral vaccination strategies, since antigen exposure alone does not elicit protective immunity. The studies are designed to increase the understanding of the mechanisms by which such adjuvants promote protective immunity, and will also generate new insights into the natural mechanisms by which the gut maintains normal immune homeostasis.